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1 Supplementary Information Facile one-pot synthesis of tetrahydroisoquinolines from amino acids via hypochlorite-mediated decarboxylation and Pictet-Spengler condensation Justin J. Maresh, Sean O. Crowe, Arthur A. Ralko, Mark D. Aparece, Casey M. Murphy, Mark Krzeszowiec, Michael W. Mullowney Contents Supporting Figures and Tables ........................................................................................................ 5 Table S1. ...................................................................................................................................... 5 Figure S1...................................................................................................................................... 5 Figure S2...................................................................................................................................... 6 Figure S3...................................................................................................................................... 6 Figure S4...................................................................................................................................... 7 Figure S5...................................................................................................................................... 7 Scheme S1. .................................................................................................................................. 8 Table S2 ....................................................................................................................................... 9 Supporting Methods ..................................................................................................................... 10 General information. ................................................................................................................ 10 Determination of sodium hypochlorite concentration ............................................................ 11 Standardization of sodium thiosulfate ................................................................................. 11 Titration of 10-15% NaOCl .................................................................................................... 11 Sodium hypochlorite oxidations ............................................................................................... 12 General Procedure for sodium hypochlorite oxidation ........................................................ 12 2 4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 12 3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 12 3-bromo-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 13 3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 13 2-phenylacetaldehyde (2e) ................................................................................................... 13 2-(1H-indol-3-yl)acetaldehyde (2f) ....................................................................................... 14 Measurement and analysis of kinetics for oxidative decarboxylation of tyrosine. .............. 14 Pictet-Spengler reactions .......................................................................................................... 15 General procedure for synthesis of racemic tetrahydroisoquinolines from α-amino acids 15 1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e) ......................................................... 17 1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f). .................................. 18 Expression of recombinant norcoclaurine synthase enzyme. .................................................. 18 General procedure for synthesis of (S)-enantiomer tetrahydroisoquinolines from α-amino acids with norcoclaurine synthase............................................................................................ 19 Norcoclaurine synthase kinetic assays for aldehyde substrates 4a – 4e. ................................ 20 Halogenation of tyrosol ............................................................................................................ 20 2-chloro-4-(2-hydroxyethyl)phenol (6b) ............................................................................... 20 2-bromo-4-(2-hydroxyethyl)phenol (6c)............................................................................... 21 2-iodo-4-(2-hydroxyethyl)phenol (6d) .................................................................................. 21 Parikh-Doering oxidation reactions .......................................................................................... 22 4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 22 3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 22 3-chloro-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 23 3 3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 23 3-Halogenation of tyrosine ....................................................................................................... 24 3-chloro-tyrosine hydrochloride (1b) ................................................................................... 24 3-bromo-tyrosine hydrochloride (1c) ................................................................................... 24 3-iodo-tyrosine (1d) .............................................................................................................. 25 Characterization Data ................................................................................................................... 26 1H NMR spectra and GC-MS data for 3-halogenated tyrosine compounds 5b-d ..................... 26 2-chloro-4-(2-hydroxyethyl)phenol (5b) ............................................................................... 26 2-bromo-4-(2-hydroxyethyl)phenol (5c)............................................................................... 27 2-iodo-4-(2-hydroxyethyl)phenol (5d) .................................................................................. 28 1H NMR spectra for 3-halogenated tyrosine analogs in D2O. ................................................... 30 3-chloro-tyrosine·HCl ............................................................................................................ 30 3-bromo-tyrosine·HCl ........................................................................................................... 30 3-iodo-tyrosine...................................................................................................................... 31 1H NMR spectra for extracted 4-hydroxyphenylacetaldehyde products (2a-2d) generated via oxidative decarboxylation of halogenated tyrosine. ................................................................ 32 4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 32 3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 32 3-bromo-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 33 3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 33 GC-MS data for 3-halogenated-4-hydroxyphenylacetaldehyde products (2a-f) generated via oxidative decarboxylation of halogenated tyrosine. ................................................................ 34 4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 34 4 3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 35 3-bromo-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 35 3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 36 1H NMR spectra for racemic tetrahydroisoquinoline products 4a-f after extraction. ............. 37 Norcoclaurine (4a) ................................................................................................................ 37 3’-chloro-norcoclaurine (4b) ................................................................................................. 38 3’-bromo-norcoclaurine (4c) ................................................................................................. 38 3’-iodo-norcoclaurine (4d) .................................................................................................... 39 1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e) ......................................................... 39 1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f) ............................... 40 References .................................................................................................................................... 40 5 Supporting Figures and Tables X H Cl Br I 6a 6b 6c 6d Yield -72 % 79 % 72 % 2a 2b 2c 2d Yield 67 % 35 % 6% 26 % Table S1. The yields of 3-halogenated-4-HPAA analogs via Parikh-Doering oxidation of tyrosol. Figure S1. Reverse phase HPLC chromatogram of a typical Parikh–Doering oxidation of tyrosol. As expected, pyridine, DMSO, and 4-hydroxyphenyl-acetaldehyde (HPAA) are present along with several other unknown side products. The plot intensity represents the maximum absorbance at each time point. Addition of pyridine has been suggested to eliminate side products that result from presence of sulfuric acid in the sulfur trioxide solution.1 We observed no significant improvement from this modification. Data obtained using Condition A (see General Information). The maximum intensity wavelength for each time is plotted. 6 Figure S2. The effect of tyrosine concentration on the efficiency of oxidative decarboxylation by NaOCl. For each reaction, a 0.1 mM solution of NaOCl was added dropwise to the tyrosine solution in 75 mM pH 7.0 phosphate buffer over 10 minutes via syringe pump at 37 °C. The undiluted reaction mixture was analyzed by reverse phase HPLC after 1.5 to 2 hours. Data obtained using Condition B (see General Information). The maximum intensity wavelength for each time is plotted. Figure S3. Time dependent behavior of oxidative decarboxylation by NaOCl. Solid lines represent a two-step irreversible kinetic model (k1 = 16 M-1s-1 and k2 = 2.6×10-3 s-1). Concentrations for intermediates are approximated by arbitrarily assigning their extinction coefficients to that of 4-HPAA. These data correspond to the experiment in Figure 1. 7 Figure S4. Regioselectivity of the reaction of 0.500 mM dopamine and 100 mM propanal with varied concentrations of phosphate buffer at pH 6.90 and 23.0 °C. The equation of the trend line is Ratio = 277×[Phosphate] + 6.05 (R² = 0.9994). Figure S5. In the presence of Tris buffer, a significant oxazolidine side product accumulates due to reaction of Tris with aldehydes. HPLC chromatogram at 280 nm of gradient 0-70% acetonitrile in 0.1 % trifluoroacetic acid (TFA) over 10 minutes monitoring a norcoclaurine synthase enzyme reaction in 100 mM TRIS at pH 7.5. Dopamine, HPAA, norcoclaurine, and 1naphthalene-acetic acid (NAA, an internal reference compound) are present along with a significant undesired oxazolidine side product (see Scheme S1). Additionally, it is apparent that Tris buffer generated numerous minor side products that are not observed in phosphate, HEPES, BES, ADA, or maleic acid buffers. 8 Scheme S1. Tris is not recommended for enzyme catalyzed tetrahydroisolquinoline synthesis as it generates oxazolidine side products that may consume as much as 50% of the aldehyde reactant, thus reducing yield and complicating isolation of tetrahydroisoquinoline products. Figure S6. Chiral HPLC chromatograms at 230 nm for norcoclaurine synthase enzyme reactions compared to reactions of the same substrates by phosphate catalyzed synthesis. Enzyme reactions A – C were carried-out in 100 mM BES buffer at pH 7.0. Enzyme reaction D was carried out in 50 mM maleic acid buffer. All data was acquired using Condition C. 9 Buffer pH of reaction Phosphate ACES ADA BES Bicine Bis-tris HEPES Imidazole Maleic acid MOPSO Tris 7.0 7.0 7.0 7.0 7.5 7.0 7.0 7.0 6.8 7.0 7.5 pKa at 25 mM and 23 °Ca 7.20 6.83 6.61 7.17 8.32 6.57 7.54 7.12 6.03 6.96 8.30 Rate of Pictet-Spengler reaction (relative to phosphate)b 100% 1.2% 4.3% 0.8% 15.4% 3.6% 0.9% 0.7% 13.2% 1.0% 7.1%c Inhibition of NCSd None 0.32 ± 0.05 None None None None 0.27 ± 0.03 0.58 ± 0.08 None 0.28 ± 0.08 None Table S2. The effects of 25 mM buffer on the relative rates of catalysis for the Pictet-Spengler reaction 2.0 mM dopamine and 2.0 mM 4-HPAA reacted in 25 mM buffer at 23 °C and inhibition of norcoclaurine synthase. a. The listed pKa values are calculated using the simplified Debye-Hückel equation described by Ellis2 for I = 0.025 M and 23 °C using the pKa values from that reference. The thermodynamic pKa2 for maleic acid was taken as 6.233. These values are given as reasonable estimates of the effective pKa. No effort was made to quantitatively control ionic strength. b. The rate of chemically-catalyzed reaction was estimated as initial rates from the linear slope the time dependent change in dopamine concentration as monitored by HPLC using Condition B (see General Information, page 10) at 225 nm. For phosphate-catalyzed data, samples were injected every 7 minutes. For other buffers, samples were injected every 14 or 21 minutes. c. Tris reacts with 4-HPAA (Scheme S1), thus the 4-HPAA concentration was not controlled. d. Inhibition of norcoclaurine synthase (NCS) in units of “percent inhibition per mM of buffer.” The initial rate was observed following the procedure for Norcoclaurine Synthase Kinetic Assays for Aldehyde Substrates (page 20). In each experiment, 20.0, 40.0, 60.0, and 80.0 mM buffer concentrations evaluated. When present, inhibition followed a linear trend. No sigmoidal trend was apparent. Uncertainty is given as the standard deviation from two replicate measurements. When “None” is stated, any change in activity from 20 to 80 mM was no greater than the detection limit (the standard deviation of the four samples). 10 Supporting Methods General information. All reagents were purchased from commercial suppliers at the highest available purity and used without further purification. Milli-Q water refers to water purified to resistivity of 18.2 MΩcm (25 °C) using an EMD Millipore Ultrapure Milli-Q reverse osmosis water purification system outfitted with ion exchange and organic removal cartridge filters. All reactions involving air or water sensitive reagents were performed using flame dried glassware under an inert atmosphere of nitrogen. Reactions were monitored using high pressure liquid chromatography on a Waters Acquity Ultra Performance Liquid Chromatography instrument with a photodiode array detector. The solvent and gradient conditions used for HPLC analysis were as follows. Condition A was used for routine analysis. Condition B provides better resolution of dopamine, aldehyde, and product peaks for kinetics and for Figure 2. Condition C was used for resolution of chiral products. Condition A: Acquity UPLC BEH C18 column (1.7 µm, 2.1x50 mm); 0.4 mL/min; 0-70% acetonitrile in 0.1 % trifluoroacetic acid (TFA) over 4.75 minutes, holding at 70% acetonitrile for 0.25 minutes. Condition B: Acquity UPLC BEH C18 column (1.7 µm, 2.1x50 mm); 0.4 mL/min, 0-17.5% acetonitrile in 0.1% TFA over 2.5 minutes followed by 17.5% to 70% acetonitrile in 0.1 % TFA over 1.5 minutes, holding at 70% for 1.0 minute. Condition C: Astec Chirobiotic™ T2 column (5 µm, 2.1x150 mm); 0.4 mL/min; isocratic mobile phase composition of 30% acetonitrile, 0.25% triethylamine, and 0.50% acetic acid in methanol. Percent conversion of reactions by HPLC was estimated from the HPLC peak areas measured at 225 nm. Area extinction coefficients for starting and product compounds were measured based on a calibration curve prepared from pure sample in water covering a concentration range from roughly 0.1 to 3000 μM. For reactions with multiple side products, the extinction coefficient was estimated by assuming that it was the same as the product. All details of UV spectra are listed from most to least intense wavelength maximum. Gas Chromatography Mass Spectroscopy analysis of products was performed using a Hewlett Packard HP6890 Gas Chromatography System with an Agilent Technologies 5975 inert mass selective detector. High resolution mass data (HRMS) were obtained on a Waters SYNAPT G1 High Definition Mass Spectrometer using an ESI ionization source. 11 1H- and 13C[1H]- NMR spectroscopy were performed using a Bruker Avance 300 MHz NMR spectrometer at 300 MHz and 75.4 MHz respectively. All 1H-NMR chemical shifts were referenced to a tetramethylsilane (TMS) internal standard. For 13C-NMR, the residual solvent peaks were used to reference the spectra as follows: in methanol-d4 was referenced as 49.00 ppm and D2O with formic acid was as 166.22 ppm. Addition of acid was required to solubilize tetrahydroisoquinoline samples. The NMR data for tetrahydroisoquinoline samples were acquired as saturated solutions. Infrared spectroscopy analysis of reaction products were performed using an ABB FT-IR FTLA 2000 spectrometer. Prepared compounds 1b-d, 2a-f, 4a, 4e, and 5b-d are known compounds, whereas 4b-d and 4f are new compounds. For new compounds, 1H-NMR, 13C-NMR, UV-Vis, and high resolution mass spectral data are provided. Determination of sodium hypochlorite concentration We found that fresh commercial solutions of NaOCl may be used by assuming the lower end of the manufacturer’s stated concentration. However, measurement of concentration appears to be essential for solutions more than six months after date of manufacture. Standardization of sodium thiosulfate.4 A solution of was prepared from 3.2 g of sodium thiosulfate pentahydrate with 0.025 g sodium carbonate (as a preservative) in 250.0 mL of MilliQ deionized water. This was standardized against a potassium iodate (KIO3) solution by dissolving approximately 2 g of potassium iodide in 5.0 mL of 6 M H2SO4 and 10.0 mL of 0.03740 M KIO3 solution. The resulting solution was titrated with the sodium thiosulfate solution until it was a pale yellow solution was observed. At this point, 1 mL of a starch indicator solution was added. The resulting blue solution was then titrated until a colorless endpoint was observed. This procedure was repeated four times yielding a concentration of (5.14 ±0.05) x 10 -2 M for the sodium thiosulfate solution. Standardized sodium thiosulfate solutions are also commercially available. Titration of 10-15% NaOCl.4 2.4476 g of a 10-15% solution of sodium hypochlorite was diluted into a 250.0 mL volumetric flask with Milli-Q water. For titration, 50.0 mL of the resulting solution was then added to a flask, with 50.0 mL of Milli-Q water, 10 mL of glacial acetic acid, and approximately 2 g of KI to yield a brown solution. This solution was titrated with standardized sodium thiosulfate until pale yellow. At this point, 1 mL of a starch indicator was added. The resulting blue solution was then titrated until the endpoint was reached, as indicated by a colorless solution. This was measurement was repeated in triplicate to determine 12 the percent of reactive NaOCl by mass. Sodium hypochlorite oxidations General Procedure for sodium hypochlorite oxidation.5, 6 To a vigorously stirring solution of amino acid (1 mmol) in 250 mL of sodium phosphate buffer (10 mM, pH 7.0) was added a solution of NaOCl (1 equivalent as a 0.1 M aqueous solution) slowly over 10 minutes via syringe pump in a 37 °C warm room or water bath. The resulting solution was stirred vigorously at this temperature until the reaction was judged to be complete by HPLC (1-2 hours). In general, the reaction may be stopped after 2 hours if monitoring is inconvenient. The solution was then extracted into dichloromethane (4 x 40 mL), washed with brine (2 x 50 mL), dried over MgSO4, and the solvent removed under reduced pressure in an unheated water bath to afford the desired aldehyde. 4-hydroxyphenylacetaldehyde (2a). DL-Tyrosine (1a, 0.200 g, 1.1 mmol), was dissolved in 250 mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 0.107 g of 2a (71 %) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 9.72 (t, J = 2.4 Hz, 1H), 7.09 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 4.82 (s, 1H), 3.62 (d, J = 2.3 Hz, 2H). LRMS (EI+)m/z 136 (M+ 17.6), 137 (M+1, 1.7), 108 (9.5), 107 (100), 89 (.63), 78 (3.7), 77 (21.6). HPLC retention time 1.63 mins condition A. Absorbance spectra λmax 222 nm minor λmax 276 nm. 3-chloro-4-hydroxyphenylacetaldehyde (2b). 3-chloro-DL-Tyrosine (0.253 g, 1 mmol), 1b, was dissolved in 250 mL of 20 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 0.143 g of 2b (83 %) as a brown oil. 1H NMR (300 MHz, CDCl3) δ 9.75 (t, J = 2.1 Hz, 1H), 7.21 (s, 1H), 7.04 (s, 2H), 5.55 (s, 1H), 3.64 (d, J = 2.1 Hz, 2H). LRMS (EI+)m/z 170 (M+, 5.89), 172 (M+2, 18.8), 143 (32.2), 141 (100), 105 (5.4), 77 (25.6). HPLC retention time 2.17 mins condition A. Absorbance spectra λmax 222 nm minor λmax 280 nm. 13 3-bromo-4-hydroxyphenylacetaldehyde (2c). 3-bromo-DL-Tyrosine (0.298 g, 1 mmol), 1c, was dissolved in 250 mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 0.177 g of 2c (82 %) as a brown crystalline semisolid. 1H NMR (300 MHz, CDCl3) δ 9.73 (t, J = 2.2 Hz, 1H), 7.33 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 8.3, 1.9 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 5.55 (s, 1H), 3.62 (d, J = 2.1 Hz, 2H). LRMS (EI+) m/z 214 (M+, 17.4), 215 (M+1, 2.5), 216 (M+2, 18.0), 188 (8.7), 187 (100), 186 (9.7), 185 (97), 107 (6.1), 106 (4.9), 78 (15.6), 77 (26). HPLC retention time 2.33 mins condition A. Absorbance spectra λmax 223 nm minor λmax 281 nm. 3-iodo-4-hydroxyphenylacetaldehyde (2d). 3-iodo-DL-Tyrosine (0.302 g, 1 mmol), 1d, was dissolved in 250 mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 0.197 g of 2d (77 %) as a brown crystalline semisolid. 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.2 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.11 (dd, J = 8.3, 2.1 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 5.36 (s, 1H), 3.63 (t, J = 3.2 Hz, 2H). LRMS (EI+) 262 (M+, 26.6), 263 (M+1, 2.2), 234 (8.4), 233 (100), 136 (0.19), 127 (2.41), 107 (3.5), 106 (16.5), 78 (10), 77 (9). HPLC retention time 2.63 mins condition A. Absorbance spectra λmax 284 nm. 2-phenylacetaldehyde (2e). DL-Phenylalanine (0.165 g, 1 mmol) was dissolved in 250 mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 72 mg of 2e (60 %) as a yellow oil. 1H-NMR, LRMS, and HPLC data matched an authentic commercial sample (Sigma-Aldrich). 14 2-(1H-indol-3-yl)acetaldehyde (2f). L-Tryptophan (205 mg, 1 mmol) was dissolved in 250 mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 45 minutes. A second equivalent of NaOCl was added and the reaction continued for a total of 2 hours to yield 118 mg of crude 2f (72%) as a brown semi-solid crude oil with impurities. HPLC analysis indicated that 35% of the tryptophan had not reacted. Additional equivalents of NaOCl resulted in the formation of significant side products. Product was not purified further. Measurement and analysis of kinetics for oxidative decarboxylation of tyrosine. The kinetics of oxidative decarboxylation were measured by quantification of HPLC peaks in a time course reaction using Condition B and observation of absorbance at 275 nm. To convert observed area to concentration, the area-extinction coefficients for starting and product compounds were measured based on a calibration curve of the pure compound dissolved in water covering a concentration range from roughly 0.1 to 3000 μM. Pure solutions of 4hydroxyphenyl-acetaldehyde (4-HPAA) were prepared by two alternative methods. By the first method, multiple solutions were prepared in volumetric glassware using the mass of pure 4HPAA oil prepared by NaOCl oxidation. By the second method, a tyrosine solutions of known concentration were reacted with NaOCl to generate 4-HPAA solutions of known concentration, assuming complete conversion (verified by HPLC). Both methods gave comparable areaextinction coefficients, however the second method generated calibration curves with significantly less deviation from linearity. The area-extinction coefficient from the latter method was used for analysis of kinetics data. A 1.00 mM solution of commercial tyrosine was prepared in 1.00 L of pH 7.0 phosphate buffer. The concentrations of phosphate buffer evaluated were 0, 3.96, 8.0, 16.0, 20.0, 40.0, and 120.0 mM. A 50 μL sample of the initial solution was removed for concentration analysis by HPLC. The solution was equilibrated with vigorous stirring at 37.0 °C in a warm room. A concentrated solution of 1.0 mmol of NaOCl was added slowly by syringe pump over 5 minutes. The change in volume resulting from this addition was less than 0.1% of the total and was treated as negligible for later rate estimates. Once addition was complete, three replicate 1 mL samples of the reaction mixture were transferred to HPLC sample vials. These vials were immediately transferred to an autosampler chamber that maintained 37.00 ± 0.01 °C for the course of the reaction. Continuous time course measurements were injected and analyzed at 7 minute 15 intervals using HPLC Condition B. As observed in Figure 2, there was no noticeable variation between replicate samples. Since three unstable intermediates were observed, it was not possible to interpolate an extinction coefficient for each of these species. Thus, the concentrations of intermediates were excluded from any analysis. The time dependent disappearance of tyrosine and appearance of 4-HPAA were fit to various models using the Gepasi computer program7. Time course curves from a two-step irreversible kinetic model most closely matched the experimental data. The rate constants for tyrosine disappearance and 4-HPAA appearance were found using a genetic algorithm (population of 10 and 1000 generations). The rates were not dependent on buffer concentration. The reported rate constant values represent the average and standard deviation from replicate measurements of all phosphate buffer concentrations from 3.96 – 40.0 mM. Samples prepared with no buffer gave inconsistent kinetic results and formed of side products. These samples were not included in the reported average. The rate of tyrosine disappearance at 120 mM phosphate buffer was slower than the other samples (5.8 ± 0.5 M-1s-1), thus data from 120 mM phosphate buffer was excluded. Pictet-Spengler reactions General procedure for synthesis of racemic tetrahydroisoquinolines from α-amino acids. Each 1 mmol of amino acid was suspended with 2.0 mL of 1.0 M phosphate buffer at pH 6.9 (this concentrated buffer gives pH 7.0 upon dilution) in 200 mL of purified water at 37 °C. A solution of 1 molar equivalent of sodium hypochlorite (e.g. 700 μL of 9.16 % NaOCl) was dissolved in 10 mL of purified water. The NaOCl was added dropwise over 10 minutes via syringe pump with vigorous stirring by magnetic stir bar. This solution was allowed to stir at 37 °C for 1.5 - 2 hours and was quenched by the addition of ascorbic acid (88 mg, 0.5 mmol). Additional phosphate catalyst, 100 mL of 1.0 M phosphate buffer at pH 6.9, was then added to the solution followed by dopamine hydrochloride (291 mg, 1.5 mmol). The reaction was monitored until all of the aldehyde was consumed as monitored by HPLC. If monitoring is not convenient, reactions are generally complete after 2 hours. The reaction was then extracted into ethyl acetate (6 X 50 mL), the combined organic layers were washed brine (2 X 50 mL), dried with MgSO4, and the solvent was removed under reduced pressure to yield the product as a solid. When scaling-up the reaction, we have found that the volume of extraction solvent can be minimized by exhaustively extracting the reaction mixture more times rather than scaling-up the extraction solvent volume. 16 Norcoclaurine (4a). DL-Tyrosine (0.189 g, 1 mmol), 3a, was reacted by the above procedure for 75 minutes to yield 0.187 g of 4a (69%) as a pale yellow solid. HPLC retention time 1.74 mins condition A. Absorbance spectra λmax 225 nm minor λmax 283 nm. 1H NMR (300 MHz, Methanold4) δ 7.10 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 8.5 Hz, 2H), 6.66 (s, 1H), 6.53 (s, 1H), 3.18 (s, 2H), 2.84 (s, 2H), 2.69 (s, 2H), 2.02 (d, J = 2.9 Hz, 1H). HRMS (C16H18NO3+): calc. 272.1287 [M+H]+; found 272.1290. 3-chloro-norcoclaurine (4b). 3-chloro-DL-Tyrosine (0.309 g, 1.5 mmol), 3b, was dissolved in 500 mL of 20 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 0.252 g of 4b (83%) as a yellow solid. HPLC retention time 1.98 mins condition A. Absorbance spectra λmax 284 nm. 1H NMR (300 MHz, methanol-d4) δ 7.25 (d, J = 2.1 Hz, 1H), 7.07 – 7.02 (m, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.64 (s, 1H), 6.56 (s, 1H), 3.29 – 3.16 (m, 3H), 2.98 (d, J = 6.7 Hz, 1H), 2.87 – 2.74 (m, 3H). 1H NMR (300 MHz, DMSO-d6) δ 8.66 (broad s, 2H), 7.24 (s, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.59 (s, 1H), 6.43 (s, 1H), 3.94 (d, J = 6.1 Hz, 1H), 3.13 – 2.89 (m, 2H), 2.89 – 2.53 (m, 3H). 13C NMR (75 MHz, D2O with 35% formic acid) δ 151.62, 144.67, 143.45, 131.40, 129.93, 128.41, 124.22, 123.39, 121.00, 117.73, 116.32, 114.24, 56.74, 39.99, 38.75, 24.60. HRMS (C16H17NO3Cl+): calc. 306.0897 [M+H]+; found 306.0910 [M+H]+. 3-bromo-norcoclaurine (4c). 3-bromo-DL-Tyrosine (0.5235, 2 mmol), 3c, was dissolved in 500 mL of 20 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 0.575 g of 4c (82%) as a tan solid. HPLC retention time 2.05 mins condition A. Absorbance spectra λmax 284 nm. 1H NMR (300 MHz, acetonitrile-d3 with 0.3% trifluoroacetic acid) δ 7.47 (d, J = 2.1 Hz, 1H), 7.13 (dd, J = 8.3, 2.1 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.67 (d, J = 4.9 Hz, 2H), 4.62 17 – 4.51 (m, 1H), 3.47 – 3.32 (m, 2H), 3.27 – 3.12 (m, 1H), 3.04 – 2.78 (m, 3H). 1H NMR (300 MHz, DMSO-d6) δ 10.20 – 9.87 (m, 1H), 8.73 (s, 1H), 8.68 – 8.50 (m, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.11 – 7.01 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.60 (s, 1H), 6.43 (s, 1H), 3.98 – 3.92 (m, 1H), 3.09 – 3.03 (m, 1H), 2.94 (s, 1H), 2.80 – 2.73 (m, 1H), 2.67 (d, J = 13.9 Hz, 1H). 13C NMR (75 MHz, methanold4 with 3% formic acid). δ 155.05, 146.86, 145.74, 135.10, 130.84, 128.92, 123.72, 123.53, 117.75, 116.23, 114.19, 111.36, 57.66, 40.88, 39.96, 25.67. HRMS (C16H17NO3Br+): calc. 350.0392 [M+H]+; found 350.0405 [M+H]+. 3-iodo-norcoclaurine (4d). 3-iodo-DL-Tyrosine (0.615, 2 mmol), 3d, was dissolved in 250 mL of 20 mM pH 7 phosphate buffer and reacted by the above procedure for 3 hours to yield 0.955 g of 4d (75%) as a yellow solid. HPLC retention time 2.21 mins condition A. Absorbance spectra λmax 285 nm. 1H NMR (300 MHz, methanol-d6 with 0.3% trifluoroacetic acid) δ 7.68 (d, J = 1.7 Hz, 1H), 7.13 (dd, J = 8.4, 1.8 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.62 (s, 1H), 6.60 (s, 1H), 4.58 (dd, J = 8.4, 5.7 Hz, 1H), 3.53 – 3.33 (m, 2H), 3.29 – 3.16 (m, 1H), 3.09 – 2.80 (m, 3H). 1H NMR (300 MHz, methanol-d6) δ 7.61 (d, J = 1.8 Hz, 1H), 7.08 (dd, J = 8.2, 1.8 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.62 (s, J = 6.6 Hz, 1H), 6.53 (s, J = 9.8 Hz, 1H), 4.12 (dd, J = 9.1, 4.3 Hz, 1H), 3.25 – 3.10 (m, 2H), 2.95 – 2.61 (m, 4H). 13C NMR (75 MHz, methanol-d4 with 6% formic acid) δ 163.39, 157.71, 146.81, 145.70, 141.24, 131.75, 129.20, 123.71, 123.52, 116.20, 114.17, 85.32, 57.74, 40.95, 39.75, 25.65. HRMS (C16H17NO3I+): calc. 398.0253 [M+H]+; found 398.0271 [M+H]+. 1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e). Prepared according to the general procedure above using L-phenylalanine (0.165 g, 1 mmol) reacted for 4 hours to yield 4e (0.149 g, 58%) as a yellow powder. 1H NMR (300 MHz, methanol-d4) δ 7.40 – 7.17 (m, 5H), 6.65 (s, 1H), 6.51 (s, 1H), 4.07 (dd, J = 9.2, 3.9 Hz, 1H), 3.29 – 3.04 (m, 2H), 2.95 – 2.75 (m, 2H), 2.74 – 2.53 (m, 2H). 1H NMR (300 MHz, DMSO-d6) δ 8.66 (broad s, 1H), 8.55 (broad s, 1H), 7.35-7.13 (m, 5H), 6.62 (s, 1H), 6.41 (s, 1H), 3.92 (d, 1H), 3.01 (d, 2H), 2.73 (t, 2H), 2.00 (s, 1H). 13C NMR (75 MHz, methanol-d4) δ 145.27, 144.71, 139.92, 130.45, 129.74, 127.67, 126.79, 116.37, 114.12, 57.95, 43.15, 41.52, 29.20. HRMS (C16H18NO2+): calc. 256.1338 [M+H]+; found 256.1324 [M+H]+; 18 additional fragments 239.1058, 161.0592. 1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f). Prepared according to the general procedure above using L-tryptophan (0.204 g, 1 mmol) and 10 mL of NaOCl solution (0.0925 M, 1 mmol). After 45 minutes of stirring, a second equivalent of 10 mL of NaOCl solution (0.0925 M, 1 mmol) was added and the reaction stirred for 90 minutes before adding ascorbic acid (88 mg, 0.5 mmol) followed by dopamine hydrochloride (291 mg, 1.5 mmol). The resulting solution was stirred for 3 hours. A dark purple powder was removed by filtration before extracting as describe to yield 4f (0.112 g, 38%) as a pale yellow solid. The yellow solid may be further purified by dissolving in minimal methanol, adding 10 volumes of chloroform, and precipitating with diethyl ether to yield a fluffy crystalline solid. The product partially overlap either the residual water or solvent peaks from all deuterated solvents in which it is soluble. Two 1H NMR spectra in different solvents were acquired for an unambiguous assignment. HPLC retention time 2.84 mins in condition A. Absorbance spectra λmax 222, 279.5 nm. 1H NMR (300 MHz, Methanol-d4) δ 7.61 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.19 (s, 1H), 7.18 – 7.10 (m, 1H), 7.06 (dd, J = 10.9, 4.0 Hz, 1H), 6.76 (s, 1H), 6.62 (s, 1H), 4.68 (dd, J = 9.2, 5.0 Hz, 1H), 3.53 – 3.39 (m, 2H), 3.28 – 3.14 (m, 2H), 2.99 – 2.83 (m, 2H). 1H NMR (300 MHz, CD3CN with 0.33% trifluoroacetic acid) δ 7.67 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.21 (s, J = 7.4 Hz, 1H), 7.26 – 7.17 (m, 1H), 7.16 – 7.08 (m, 1H), 6.89 (s, 1H), 6.69 (s, 1H), 4.79 – 4.63 (m, 1H), 3.63 (dd, J = 15.3, 4.5 Hz, 1H), 3.50 – 3.37 (m, 1H), 3.27 – 3.11 (m, 2H), 3.01 – 2.81 (m, 2H). 13C NMR (75 MHz, methanol-d with 1.5% formic acid) δ 146.82, 145.91, 138.50, 128.23, 4 125.59, 124.14, 123.70, 123.04, 120.36, 119.12, 118.17, 116.21, 114.11, 112.72, 108.81, 56.68, 40.85, 31.42, 25.71. HRMS (C18H19N2O2+): calc. 295.1447 [M+H]+; found 295.1429 [M+H]+; major fragment 164.0698. Expression of recombinant norcoclaurine synthase enzyme. We followed a modification of the procedure described by Luk et al.9 except using the plasmid construct of Ruff et al.10 This plasmid contains a codon-optimized sequence of TfNCSΔ19 (5071-5769), Thalictrum flavum norcoclaurine synthase with the N-terminal 19 amino acids deleted. This sequence was cloned into the pET-28a(+) vector between the NdeI and EcoRI restriction sites to generate plasmid pNCSa. This plasmid was transformed into BL21(DE3) chemically competent cells (Sigma). 19 The cells were incubated with shaking at 250 rpm in 50 mL of Luria-Bertani (LB) medium containing BL21(DE3)/pNCSa and 30 μg/mL kanamycin overnight at 37 °C. The overnight cultures were poured into 1.0 L of LB medium containing 30 μg/mL kanamycin and grown at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. Cells were induced for overexpression by addition of 238 mg (1 mM) of isopropyl-β-D-galactopyranoside (IPTG), and the cultures were allowed to continue growth at 25 °C until an OD600 of 3.0 - 4.0 was reached (~24 h). Cells were harvested by centrifugation at 4000 rcf for 20 min and resuspended in binding buffer (50 mM phosphate, 20 mM imidazole, 300 mM NaCl at pH 8.0). The cells were lysed by sonication at 250 Watts for 120 secs in cycles of 10 seconds on and 10 seconds of cool down. The cell lysate was clarified by centrifugation at 20,000 rcf for 25 min. The supernatant was drawn through a 10 mL syringe with 22 gauge needle 80 times to shear DNA and filtered through a 0.22 μm membrane filter. A 1 mL GE Healthcare HisTrap FF nickel affinity column was washed with 10 column volumes (CV) of water, 10 CV of binding buffer (50 mM phosphate, 20 mM imidazole, 300 mM NaCl at pH 8.0) to prime the column. The filtered cell lysate was loaded at a rate of 1 mL/min. Binding buffer (10 CV) was passed through the column at 1 mL/min. A wash with wash buffer (20 mM imidazole, 500 mM NaCl at pH 7.8) was used to remove nonspecifically bound proteins until no more flow-through protein eluted, as determined by measurement of A280 (~50 CV). Histidine-tagged protein was eluted with 21 CV of elution buffer (300 mM imidazole and 500 mM NaCl at pH 7.0). The enzyme was concentrated by ultrafiltration (Amicon Ultra-4, 10 000 MWCO) to a volume of 500 μL. This solution diluted into equal volume glycerol to yield 3 -7 mg/mL as determined by Bradford assay. The solution was divided into 500 μL aliquots and stored at -20 °C without freezing and with no noticeable loss of activity after several months. The enzyme was not allowed to warm before use. General procedure for synthesis of (S)-enantiomer tetrahydroisoquinolines from α-amino acids with norcoclaurine synthase. Each 1 mmol of amino acid was suspended in 250 mL of 25 mM pH 6.80 maleic acid buffer at 37 °C. A solution of 1 molar equivalent of sodium hypochlorite (e.g. 700 μL of 9.16 % NaOCl) was dissolved in 10 mL of purified water. The NaOCl was added dropwise over 10 minutes via syringe pump with vigorous stirring by magnetic stir bar. This solution was allowed to stir at 37 °C for 1.5 – 2 hours and was quenched by the addition of ascorbic acid (88 mg, 0.5 mmol). After 5 minutes, 1.5 molar equivalents of dopamine hydrochloride (291 mg, 1.5 mmol) was added followed by 2.5 mg of norcoclaurine synthase (isolated by the procedure above). After stirring for 2 hours at 37 °C, the reaction mixture was analyzed. HR-MS and HPLC-UV properties were identical to racemic norcoclaurine synthesized in phosphate buffer. Chiral HPLC revealed only one detectable enantiomer. All other characterization data were identical to the equivalent enantiomeric mixture prepared in phosphate buffer. 20 Norcoclaurine synthase kinetic assays for aldehyde substrates 4a – 4e. The initial rate of enzyme reaction was measured for substrates 4a – 4e by preparing a 500 μL reaction containing a final concentration of 1.00 mM dopamine HCl (from 10.0 mM stock solution in deionized water), 1.00 mM aldehyde (from 100 mM DMSO stock solution), 100 mM HEPES (or BES, both gave identical results) at pH 7.2, and 10 μg of norcoclaurine synthase enzyme (prepared as described above). For the total number of aldehyde samples, all other components of the reaction except for the aldehyde (dopamine, buffer, and enzyme) were mixed as a single stock solution and then divided into equal aliquots. The reaction was started by the addition of aldehyde. The reactions were quenched every 60 seconds by removing 80 µL of the reaction and mixing it with 20 µL of 1 M HCl. The rate of the reaction was determined by monitoring the area of the dopamine peak by HPLC (condition B) at 280 nm. The resulting areas were then plotted as a function of time to yield straight lines, the slopes of which gave the rates in units of µV*s /s. All rates are reported as a percent of the rate of positive control 4a within the same experiment. All analyses were repeated three independent times under identical conditions. Halogenation of tyrosol 2-chloro-4-(2-hydroxyethyl)phenol (6b).11 A suspension containing 4-(2-hydroxyethyl)phenol (6a, 2.990, 21.6 mmol) and NaCl (1.266 g, 21.6 mmol) in acetone (55 mL) was cooled to 0 oC while a solution of Oxone (19.818 g, 32.2 mmol) in water (100 mL) was added dropwise over three hours. The reaction was then allowed to stir overnight at room temperature, after which the reaction was extracted with ethyl acetate (3 x 60 mL) and the combined organic layers washed with brine (1 x 30 mL). The resulting solution was then dried with MgSO 4, filtered and the solvent removed under reduced pressure yielding an amber colored oil. The resulting oil was then dissolved in a minimal amount of hot dichloromethane and cooled yielding 3.735 g of 6b (72 %) as white crystals. 1H NMR (300 MHz, Acetone-d6) δ 8.64 (s, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.2, 2.1 Hz, 1H), 6.94 – 6.88 (m, 1H), 3.73 – 3.70 (m, 2H), 2.74 – 2.69 (m, 2H). LRMS (EI+)m/z 172 (M+, 25), 173 (M+1, 2,23), 174 (M+2, 8.4), 143 (33.2), 142 (9.5), 141 (100), 107 (5.4), 105 (4.5), 77 (19.3). HPLC retention time 2.18 mins condition A. Absorbance spectra λmax 222 nm minor λmax 280 nm. 21 2-bromo-4-(2-hydroxyethyl)phenol (6c).11 A suspension containing 4-(2-hydroxyethyl)phenol (6a, 3.463, 25 mmol) and NaBr (2.589 g, 25.2 mmol) in acetone (55 mL) was cooled to 0 °C while a solution of Oxone (23.153 g, 32.2 mmol) in water (100 mL) was added dropwise over three hours. The reaction was then allowed to stir for three additional hours at room temperature, after which the reaction was extracted with ethyl acetate (3 x 60 mL) and the combined organic layers washed with brine (1 x 30 mL). The resulting solution was then dried with MgSO 4, filtered and the solvent removed under reduced pressure yielding an amber colored oil. The resulting oil was then dissolved in a minimal amount of hot dichloromethane and cooled yielding 4.292 g of 6c (79 %) as a white solid 1H NMR (300 MHz, Acetone D-6) δ 8.64 (s, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.07 (dt, J = 8.1, 4.1 Hz, 1H), 6.95 – 6.89 (m, 1H), 3.72 (dd, J = 6.1, 4.4 Hz, 2H), 2.79 – 2.66 (m, 2H). LRMS (EI+)m/z 216 (M+, 23.8), 218 (M+2, 24.3), 173 (16), 188 (9.0), 187 (97.9), 186 (9.1), 185 (100), 107 (10) 106 (4.4) 78 (9.3), 77 (21.7). HPLC retention time 2.31 mins. Absorbance spectra λmax 218 nm minor λmax 281 nm. 2-iodo-4-(2-hydroxyethyl)phenol (6d).12 A solution of I2 (5.074 g, 20 mmol) and KI (4.374 g, 26 mmol) in 100 mL of a 1:1 water and ethanol was added to a solution containing 4-(2hydroxyethyl)phenol (6a, 2.733 g, 20 mmol) dissolved in 20 mL of a 40% solution of dimethyl ammine. The resulting solution was allowed to stir at root temp for 4 hours after which it was acidified with a 2 M solution a HCl and extracted into ethyl acetate (3x100 mL). The combined organic layers were then washed with a 10% (m/V) solution of sodium thiosulfate (2x50 mL) and brine (1x40mL). The resulting organic layer was then dried with MgSO4, filtered, and the solvent removed under reduced pressure yielding a brown oil. The resulting oil was dissolved in a minimal amount of hot water and cooled yielding 3.7602 g of 6d (72 %) as white needle like crystals. 1HNMR (300 MHz, CDCl3) δ 7.55 (d, J = 2.0 Hz, 2H), 7.12 (dd, J = 8.2, 2.0 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 5.40 (s, 2H), 3.84 (t, J = 6.5 Hz, 5H), 2.84 – 2.63 (m, 5H), 1.60 (d, J = 33.9 Hz, 4H). LRMS (EI+) m/z 264 (M+ 35.2), 265 (M+1 3.0), 234 (9.8), 233 (100), 128 (1.4), 107 (5.0), 106 (13.9) 78 (7.8), 77 (6.5). HPLC retention time 2.48 mins condition A. Absorbance spectra λmax 217 nm minor λmax 284 nm. 22 Parikh-Doering oxidation reactions 4-hydroxyphenylacetaldehyde (2a).1, 8, 13-15 In a flame dried flask purged with N2, sulfur trioxide pyridine complex (3.114 g, 20 mmol) was dissolved in 15 mL of dimethyl sulfoxide dried over 3A molecular sieves.16 This solution was then transferred by cannula into a second flame dried flask purged with N2 containing 4-(2-hydroxyethyl)phenol (6a, 0.951 g, 6.8 mmol) and triethylamine (3 mL, 21 mmol), dissolved in 15 mL of sieve dried dimethyl sulfoxide, slowly over a period of 15 mins. The resulting solution was allowed to stir at room temperature for 1.5 hours under a N2 atmosphere, after which it was poured into 100 mL of ice cold water and acidified with 10 mL of 2 M HCl. The resulting solution was extracted into ethyl acetate (3x100 mL), and the combined organic fractions washed with brine (2x100 mL), dried with MgSO 4, filtered and the solvent removed under reduced pressure yielding a crude amber colored oil. The oil was purified by flash chromatography (20% ethyl acetate in hexanes) yielding 0.2272g of 2a (24%) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.4 Hz, 1H), 7.15 – 7.06 (m, 2H), 6.90 – 6.82 (m, 2H), 4.80 (s, 1H), 3.63 (dd, J = 8.4, 2.9 Hz, 2H). LRMS (EI+) m/z 136 (M+ 17.6), 137 (M+1, 1.7), 108 (9.5), 107 (100), 89 (.63), 78 (3.7), 77 (21.6). HPLC retention time 1.63 mins condition A. Absorbance spectra λmax 222 nm minor λmax 276 nm. 3-chloro-4-hydroxyphenylacetaldehyde (2b). In a flame dried flask purged with N2, sulfur trioxide pyridine complex (0.311 g, 1.9 mmol) was dissolved in 5 mL of dimethyl sulfoxide dried over 3A molecular sieves.16 This solution was then transferred by cannula into a second flame dried flask purged with N2 containing 6b (0.129 g, 0.75 mmol) and triethylamine (0.33 mL, 3.7 mmol), dissolved in 5 mL of sieve dried dimethyl sulfoxide, slowly over a period of 15 mins. The resulting solution was allowed to stir at room temperature for 4 hours under a N 2 atmosphere, after which it was poured into 100 mL of ice cold water. The resulting solution was extracted into ethyl acetate (3x50 mL), and the combined organic fractions washed with brine (1x40 mL), dried with MgSO4, filtered and the solvent removed under reduced pressure yielding a crude amber colored oil. Flash chromatography was performed (0-40% ethyl acetate in hexanes) yielding 0.127 g of 2b (35.5%). 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.2 Hz, 1H), 7.20 (d, J = 6.4 23 Hz, 1H), 7.12 – 7.01 (m, 2H), 5.62 (s, 1H), 3.66 (t, J = 11.0 Hz, 2H). LRMS (EI+)m/z 170 (M+, 5.89), 172 (M+2, 18.8), 143 (32.2), 141 (100), 105 (5.4), 77 (25.6). HPLC retention time 2.17 mins condition A. Absorbance spectra λmax 222 nm minor λmax 280 nm. 3-chloro-4-hydroxyphenylacetaldehyde (2c). In a flame dried flask purged with N2, sulfur trioxide pyridine complex (0.897 g, 5.6 mmol) was dissolved in 10 mL of of dimethyl sulfoxide dried over 3A molecular sieves.16 This solution was then transferred by cannula into a second flame dried flask purged with N2 containing 6c (0.5858 g, 2.7 mmol) and triethyl amine (0.75 mL, 5.4 mmol), dissolved in 10 mL of sieve dried dimethyl sulfoxide, slowly over a period of 15 mins. The resulting solution was allowed to stir at room temperature for 1 hour under a N 2 atmosphere, after which it was poured into 150 mL of ice cold water. The resulting solution was extracted into ethyl acetate (3x75 mL), and the combined organic fractions washed with brine (1x50 mL), dried with MgSO4, filtered and the solvent removed under reduced pressure yielding a crude orange oil. Flash chromatography was performed (25% ethyl acetate in hexanes) yielding 0.0361 g of 2c (6.2%). 1H NMR (300 MHz, CDCl3) δ 9.72 (d, J = 16.0 Hz, 1H), 7.35 (s, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 5.61 (s, 1H), 3.61 (d, J = 22.4 Hz, 2H). LRMS (EI+) m/z 214 (M+, 17.4), 215 (M+1, 2.5), 216 (M+2, 18.0), 188 (8.7), 187 (100), 186 (9.7), 185 (97), 107 (6.1), 106 (4.9), 78 (15.6), 77 (26). HPLC retention time 2.33 mins condition A. Absorbance spectra λmax 223 nm minor λmax 281 nm. 3-iodo-4-hydroxyphenylacetaldehyde (2d). In a flame dried flask purged with N2, sulfur trioxide pyridine complex (0.869 g, 5.5 mmol) was dissolved in 10 mL of dimethyl sulfoxide dried over 3A molecular sieves.16 This solution was then transferred by cannula into a second flame dried flask purged with N2 containing 6d (0.7060 g, 2.7 mmol) and triethylamine (0.75 mL, 5.4 mmol), dissolved in 10 mL of sieve dried dimethyl sulfoxide, slowly over a period of 15 mins. The resulting solution was allowed to stir at room temperature for 1 hour under a N 2 atmosphere, after which it was poured into 100 mL of ice cold water. The resulting solution was extracted into ethyl acetate (3x80 mL), and the combined organic fractions washed with brine (1x100 24 mL), dried with MgSO4, filtered and the solvent removed under reduced pressure yielding a crude orange oil. Flash chromatography was performed (30% ethyl acetate in hexanes) yielding 0.1804 g of 2d (26%) as a slightly colored oil. 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.2 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.12 (dd, J = 8.3, 2.1 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 5.29 (s, 1H), 3.63 (d, J = 2.2 Hz, 2H). LRMS (EI+) 262 (M+, 26.6), 263 (M+1, 2.2), 234 (8.4), 233 (100), 136 (0.19), 127 (2.41), 107 (3.5), 106 (16.5), 78 (10), 77 (9). HPLC retention time 2.63 mins condition A. Absorbance spectra λmax 284 nm. 3-Halogenation of tyrosine 3-chloro-tyrosine hydrochloride (1b).17 Tyrosine (1a, 7.567 g, 41.8 mmol) was suspended in 75 mL of glacial acetic acid and SO2Cl2 (3.44 mL, 44 mmol) was slowly added to the suspension over 10 minutes. The resulting solution was allowed to stir for 5.5 hours, after which the solid precipitate was collected by vacuum filtration and washed with glacial acetic acid (3x10 mL) and diethyl ether (3x25mL). The resulting product was then recrystallized from boiling concentrated hydrochloric acid and dried under vacuum to yield 6.910 g of 1b (66%) as white crystals. 1H NMR (300 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.25 (s, 3H), 7.24 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.3, 2.1 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 4.14 (s, 1H), 3.00 (t, J = 5.7 Hz, 2H). HPLC retention time 1.56 mins condition A. Absorbance spectra λmax 222 nm minor λmax 280 nm. 3-bromo-tyrosine hydrochloride (1c).18 A solution containing tyrosine (1a, 5.541 g, 30.6 mmol) suspend in 25 mL of glacial acetic acid and 15 mL of a 33% solution of HBr in acetic acid (61 mmol), was prepared. A solution of bromine (1.7 mL, 33.2 mmol) in 25 mL of glacial acetic acid was then added to the previously prepared solution over a period of 3 hours, and the resulting solution was allowed to stir for 24 hours. Upon completion of the reaction the solid precipitate was collected by vacuum filtration, washed with glacial acetic acid (3x25 mL) and diethyl ether 25 (3x25mL) yielding an orange powder. The powder was recrystallized from boiling concentrated hydrochloric acid and dried under vacuum to yield 6.023g of 1c (70%) as white crystals. 1HNMR (300 MHz, D2O) δ 7.40 (d, J = 2.1 Hz, 1H), 7.06 (dd, J = 8.3, 2.1 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 4.12 (d, J = 5.4 Hz, 1H), 3.15 (dd, J = 14.7, 5.6 Hz, 1H), 3.02 (dd, J = 14.8, 7.6 Hz, 1H). HPLC retention time 1.64 mins condition A. Absorbance spectra λmax 280 nm. 3-iodo-tyrosine (1d).19 A solution of 1 (4.968 g, 27 mmol) dissolved in concentrated NH4OH was cooled in an ice water bath, and iodine (7.0325 g, 27.8 mmol)dissolved in 150 mL of ethanol was slowly added dropwise via syringe pump. The resulting solution was allowed to stir overnight, after which the solvent was removed under reduced pressure, yielding an off white solid, which was suspended in 150 mL of ice cold water. The solid was collected by vacuum filtration and re-suspended in ice cold acetone and stirred for 1.5 hours. The resulting solid was collected by vacuum filtration yielding 2.528 g of 1d (30 %) as a white powder. 1H NMR (300 MHz, D2O) δ 7.60 (d, J = 2.1 Hz, 1H), 7.12 – 7.03 (m, 1H), 6.83 (d, J = 8.3 Hz, 1H), 3.84 – 3.78 (m, 1H), 3.10 – 3.01 (m, 1H), 2.91 (dd, J = 14.7, 7.8 Hz, 1H). HPLC retention time 1.78 mins condition A. Absorbance spectra λmax 283 nm. 26 Characterization Data 1H NMR spectra and GC-MS data for 3-halogenated tyrosine compounds 5b-d 2-chloro-4-(2-hydroxyethyl)phenol (5b) 27 2-bromo-4-(2-hydroxyethyl)phenol (5c) 28 2-iodo-4-(2-hydroxyethyl)phenol (5d) 29 30 1H NMR spectra for 3-halogenated tyrosine analogs in D2O. 3-chloro-tyrosine·HCl 3-bromo-tyrosine·HCl 31 3-iodo-tyrosine 32 1H NMR spectra for extracted 4-hydroxyphenylacetaldehyde products (2a-2d) generated via oxidative decarboxylation of halogenated tyrosine. 4-hydroxyphenylacetaldehyde (2a) 3-chloro-4-hydroxyphenylacetaldehyde (2b) 33 3-bromo-4-hydroxyphenylacetaldehyde (2c) 3-iodo-4-hydroxyphenylacetaldehyde (2d) 34 GC-MS data for 3-halogenated-4-hydroxyphenylacetaldehyde products (2a-f) generated via oxidative decarboxylation of halogenated tyrosine. 4-hydroxyphenylacetaldehyde (2a). Expect M+ 136.15 Da. 35 3-chloro-4-hydroxyphenylacetaldehyde (2b). Expect M+ 170.01 and (M+2)+ 172.02 Da 3-bromo-4-hydroxyphenylacetaldehyde (2c). Expect M+ 213.96 and (M+2)+ 215.96 Da. 36 3-iodo-4-hydroxyphenylacetaldehyde (2d). Expect M+ 262.04 Da. 37 1H NMR spectra for racemic tetrahydroisoquinoline products 4a-f after extraction. Norcoclaurine (4a). Solvent peaks at 4.8 and 3.6 ppm are water and methanol-d4. 38 3’-chloro-norcoclaurine (4b). 3’-bromo-norcoclaurine (4c). Solvent peaks at 1.94 and 5.10 ppm are residual acetonitrile-d3 and TFA (added to promote solubility). Phenol protons appear in the aromatic region under these conditions. 39 3’-iodo-norcoclaurine (4d). Solvent peaks at 4.8 and 3.6 ppm are water and methanol-d4. The peaks at 1.22 (t), 2.03 (s), and 4.06 (q) are residual ethyl acetate. 1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e). Solvent peaks at 4.85, 3.31, and 3.34 ppm are water, methanol-d4, and methanol. 40 1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f). Solvent peaks at 4.85, and 3.31 ppm are water and residual methanol-d4 respectively. 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